The expedition comes despite an International Court of Justice ruling and fierce objections from countries including Australia and New Zealand.
"We do not accept in any way, shape or form the concept of killing whales for so-called 'scientific research," said Australian Environment Minister Greg Hunt.
New Zealand's acting Foreign Minister Todd McClay said: "New Zealand is strongly opposed to whaling in the Southern Ocean. We call on Japan to take heed of the 2014 International Court of Justice decision and international scientific advice concerning their whaling activities."
Japan has consistently rejected calls for its to cease its whaling activities, insisting that it's vital for building a body of research.
"Through capturing whales for investigation, Japan is collecting the scientific data and aiming for the resumption of commercial whaling. This official government view doesn't change," Hideki Moronuki, senior fisheries negotiator at Japan's Fishery Agency told CNN.
A fleet of four vessels left the port of Shimonoseki, southwest of Tokyo, on Tuesday, for the Southern Ocean, including research ship the "Nisshin Maru" and three smaller boats. The expedition is due to end in March 2016.
Court issues stop order
Last March, the International Court of Justice ordered Japan to halt its JARPA II research program,
after rejecting the country's claims that it was for scientific purposes.
"Japan shall revoke any extant authorization, permit or license granted in relation to JARPA II, and refrain from granting any further permits in pursuance of that program," the court said.
Japan said the purpose of JARPA II was to monitor the Antarctic ecosystem, model competition among whale species and improve the management of minke whale stocks.
Japan hunts whales despite a worldwide moratorium, taking advantage of a loophole in the law that permits the killing of the mammals for scientific research. Whale meat is commonly available for consumption in Japan.
Researchers or poachers?
After the court ruling, Japan announced a new research program with a new name -- NEWREP-A
-- which is scheduled to last 12 years, until 2026/27.
Under the program, a maximum Antarctic 333 minke whales will be killed each year, according to
an information sheet from Japan's Fisheries Agency and Ministry of Foreign Affairs.
That's almost 4,000 over the duration of the program.
In justifying the slaughter, the sheet says: "As there is no other means than lethal methods, at this stage, the use of lethal method is indispensable to obtain age data which is necessary for estimating the age-at-sexual maturity (ASM), which makes considerable contribution to achieving the application of the RMP (Research Management Procedure).
Conservation group Sea Shepherd says the Japanese are merely poachers who are breaking international law.
"We would like to remind the Japanese government that the whales of the Southern Ocean are protected by international law, by Australian law and by Sea Shepherd," Sea Shepherd's CEO, Captain Alex Cornelissen in a statement.
As such, any violation of the sanctity of the Southern Ocean Whale Sanctuary or the Australian Whale Sanctuary will be regarded as a criminal act," he added.
The group has called on the Australian government to defend its waters, and the chair of Sea Shepherd's Australian board has suggested that the group will intervene if necessary.
"If Sea Shepherd comes across criminal activity, then our history speaks for itself. We will, as always, directly intervene to prevent that crime from taking place," said Captain Peter Hammardstedt.